Endotoxin, or lipopolysaccharide, is a component of the cell wall of Gram-negative bacteria and is implicated in the manifestation of acute bacterial infections. Numerous proteins have been reported which bind to the principal form of endotoxin, lipopolysaccharide ("LPS"). Examples of such LPS-binding proteins are bactericidal/permeability-increasing protein ("BPI protein") lipopolysaccharide-binding protein ("LBP"), incorporated by reference herein], high density lipoprotein, and tachyplesin [Nakamura, et al., J. Biol. Chem., 263:16709-16713 (1988), incorporated by reference herein].
Certain of these proteins share significant structural homology. For example, both BPI and LBP possess a positively-charged amino terminal region of approximately 25 kDa which is the portion of each molecule which binds to the lipid A portion of LPS. See Schumann, et al., Science, 249:1429-1431 (1990).
Binding of BPI protein to membrane-bound LPS increases the envelope permeability of susceptible Gram negative bacteria. Ooi, et al., J. Biol. Chem., 262:14891 (1987). BPI protein also binds to soluble LPS and human BPI protein has been isolated from polymorphonuclear neutrophils ("PMNs") by acid extraction combined with either ion exchange chromatography or E. coli affinity chromatography. Elsbach, et al. J. Biol. Chem., 254:11000 (1979); Weiss et al., Blood, 69:652 (1987). The holo-BPI protein isolated from human PMNs has potent bactericidal activity against a broad spectrum of Gram-negative bacteria. Elsbach, et al., J. Biol. Chem., 254:11000 (1979). This antibacterial activity appears to be associated with the amino terminal region of the isolated human holo-BPI protein. In contrast, the carboxyl terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi, et al., J. Exp. Med., 174:649 (1991).
Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated [See, Gray et al., J. Biol. Chem., 264:9505 (1989), hereinafter referred to as "Gray"; U.S. Letters Pat. No. 5,198,541, both of which are incorporated by reference herein] allowing for the large scale production of recombinant BPI and biologically active (e.g., amino and carboxyl terminal) fragments thereof. Initial attempts to purify recombinant BPI and BPI-related proteins from the medium of transfected cells utilizing traditional protein purification methods provided low yields. Pulse-chase experiments using .sup.35 S labelled methionine and performed on cell cultures of transfected Chinese Hamster Ovary (CHO) cells expressing a recombinant product comprising the amino terminal 199 amino acids of the mature BPI protein [hereinafter rBPI(1-199)] indicated that the recombinant BPI fragment disappeared from the media during 3.5 hours to 7 hours of chase. Preliminary experimental procedures aimed at determining the basis for this low product yield indicated that the protein product displays significant "stickiness" and, in fact, adheres to itself, to other media components (including host cells), and to plastic and glass culture vessels. However, the precise reason(s) for protein loss have yet to be elucidated.
Like BPI protein, LBP binds to the lipid A portion of LPS. The holo-LBP protein is a 60 kD protein secreted by the liver and has been reported to be responsible for delivering LPS to macrophages. Ooi, et al., J. Exp. Med., 174:649-65 (1991). Unlike BPI protein, LBP generally enhances the inflammatory response generated by LPS. For example, LBP stimulates LPS-induced tumor necrosis factor ("TNF") production.
Of interest to the present invention is the use of ion exchange materials in the isolation and purification of proteins and related substances. For example, published PCT application No. WO 89/05157 by Prior, et al., reports the purification and isolation of recombinant immunoglobulins by passing the cell culture medium over a chromatography column, wherein the immunoglobulin is adsorbed onto an exchange material. The immunogiobulin is then eluted by raising the salt concentration in the column. As another example, published PCT application No. WO 90/08159 by Robins, et al., reports removal of DNA from protein preparations by incubation in the presence of an anion exchange material. Wang, Ann. N.Y. Acad. Sci., 413:313-321 (1983) presents the results of "hybrid" fermentation-extraction procedures for the production and isolation of a model antibiotic, cycloheximide, from fermentation cultures using non-ionic resins and noted that for one resin (XAD-4, Rohm and Haas, Philadelphia, Pa.) the product was absorbed on the resin surface making it "conceivable" to harvest the product from the resin.
Due to the utility of endotoxin binding proteins such as BPI protein and LBP as regulators of bacterial infection and the sequelae thereof, there exists a need in the art for improved methods for the isolation of such proteins from cell culture media.